We will pursue a series of closely related projects in order to characterize the nature and interrelationships of repair, recombination and meiosis in the yeast Saccharomyces cerevisiae. The central questions will concern the exact role o DNA repair (RAD) genes in normal cell metabolism, including mitotic and meiotic recombination, as well as how they function in DNA repair. We will study those RAD genes whose mutants confer sensitivity to X-rays, since it is primarily these mutants which show complex phenotypes involving normal mitotic or meiotic DNA metabolism as well as repair. We will study both new and currently available X-ray sensitive mutants, focusing on three areas: (1) Genetic Studies: Recently isolated rad mutations will be examined to see if they define new RAD loci and, if so, will be mapped and characterized for their repair, recombination and meiotic phenotypes. To determine if any of the existing RAD genes code for essential functions, we will isolate both deletions and nonsense alleles by in vitro mutagenesis with cloned genes. Reversion studies on some of the rad mutations will be done to isolate new temperature conditional and regulatory alleles, and to determine if the RAD gene products interact with each other or other proteins. We will continue our studies on chromosome loss in existing rad mutants. (2) Meiotic Studies: RAD genes whose mutant alleles are defective in sporulation are likely to control aspects of meiotic DNA metabolism, and we therefore plan to study their meiotic defects to learn more about both the RAD gene products and the process of meiosis. We will perform temperature-shifts on double-mutant strains containing heat-sensitive and cold-sensitive alleles. We will also study strains containing rad mutants in combination with meiosis-I by-pass mutations. These approaches in combination with transcriptional studies will provide information about dependent sequences and the order of gene functions in meiosis, as well as the timing of various stages of recombination and their relationship to other aspects of the meiotic divisions. (3) Molecular Studies: Using already isolated clones of six RAD genes, we will study various aspects of the transcriptional regulation of these genes. We will determine if and when these genes are transcribed during meiosis and whether they are induced by DNA damaging agents in mitotic cells.